Cartilage is an avascular connective tissue, distributed in all parts of the body. It consists of cartilage cells and extracellular matrix which is secreted and maintained by cartilage cells. According to the morphological criteria and the content of collagen and elastin, cartilage can be divided into three categories: hyaline cartilage, such as the nasal septum, articular cartilage, trachea and bronchi; elastic cartilage, such as ear and epiglottis; and fibrocartilage, such as meniscal cartilage and intervertebral disc. Naumann, A, etc. published a report entitled “Immunochemical and mechanical characterization of cartilage subtypes in rabbit” in the “Journal of Histochemistry and Cytochemistry” (2002(50):1049-1058), which comments that hyaline cartilage contains mostly type II collagen, elastic cartilage contains mainly elastin and fibrocartilage contains mostly type I collagen.
Articular cartilage and the intervertebral disc play an important role in the systemic motion. The homeostasis of extracellular matrix of articular cartilage and intervertebral disc depends on their anabolic and catabolic metabolism in articular cartilage and intervertebral disc. When the balance of cartilage matrix breaks, cartilage-matrix gradually loses its normal physiological functions, eventually leading to joint and disc disease, such as osteoarthritis and intervertebral disc degeneration. As the cartilage has very limited ability to repair itself, a variety of clinical methods and experimental methods which are used to improve the cartilage regeneration can not efficiently repair the damaged cartilage to meet clinical needs.
Biological treatment of damaged cartilage may replace chronic traditional treatment methods and reconstruction surgical methods. However, these biological treatment methods rely on the use of growth factors. At present, many cytokines are in development, specifically for cell growth, differentiation and cartilage matrix synthesis, and so on. These include fibroblast growth factors (FGFs), insulin-like growth factor-1 (IGF-1), transforming growth factor-β (TGF-β) cartilage-derived morphogenetic proteins (CDMPs), bone morphogenetic proteins (BMPs) and connective tissue growth factor (CCN2/CTGF). Shida, J, etc. published a report entitled “Regulation of osteoblast, chondrocyte and osteoclast functions by fibroblast growth factor (FGF)-18 in comparison with FGF-2 and FGF-10” in the “Journal of Orthopaedic Research” (2002(277):493-7500). It comments that when FGF-2 and FGF-18 are given by intra-articular injection, FGF-2 can not only promote the increase of the articular cartilage of the young rats, but also promote the proliferation of mesenchymal cells and cover the articular surface. Ellsworth, J. L, etc. published a report entitled “Fibroblast growth factor (FGF)-18 is a trophic factor for mature chondrocyte and their progenitors” in the “Osteoarthritis and Cartilage” (2002(10):308-320). It comments that expression of FGF-18 by adenovirus promoted the formation of cartilage cells around the virus injection site, but had no effect on the mouse ear elastic cartilage. Therefore, there is a need to discover a growth factor which can promote the growth of cartilage tissue in vivo, rather than promote the proliferation of other cell types.
Cartilage tissue engineering is a technology used for cartilage disease and injury. Autologous chondrocyte transplantation (ACT) technology has been used in clinical treatment for craniofacial and joint cartilage damage. So far, ACT technology has cured more than 12,000 patients with full-thickness cartilage defects worldwide. The main challenge of cartilage tissue engineering is to obtain sufficient cartilages cells to fill the cartilage defect site. Due to the limited number of cartilage cells in vivo, only 5% to 10% of chondrocytes in cartilage tissue, in vitro expansion is required before clinical use. Brittberg, M, etc. published a report entitled “Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation” in the “New England Journal of Medicine” (1994(331):889-895). It comments that chondrocyte are mainly isolated from hyaline cartilage of joint and elastic cartilage of ear, and cultured in vitro. Currently chondrocytes are cultured in vitro in monolayer with serum-containing medium. Although this method is very effective in expansion of chondrocytes, chondrocytes often differentiate into fibroblasts and lose the chondrocyte characteristics. The first to fourth passages of subcultured chondrocytes maintain their chondrocyte characteristics when they are cultured in high-density or transplanted into animals (this process is known as re-differentiation). Therefore, the ability of re-differentiation is closely related to the number of passages in culture. At present, a variety of growth factors such as FGF-2, TGF-β, BMP-2 and IGF-1 have been applied to chondrocyte culture in vitro for promoting chondrocyte proliferation, reducing differentiation and re-differentiation. Currently, it is an important goal to search for an ideal chondrocyte growth factor for chondrocyte culture in vitro. This ideal growth factor can promote the amplification of three kinds of cartilage cells and maintain their characteristic gene expression profiles and the synthesis of the cartilage-specific matrix in limited subculture.
Midkine (MK) is a member of pleiotropic factor/midkine family, it was originally described as a factor that is produced by retinoic acid, and heparin-binding neurotrophic factor which regulates growth. MK is highly expressed in the brain and other tissue in the second trimester, and decreased after birth. In adult animals, MK is strictly expressed with high level of transcription in the small intestine, and low expression in other tissues. Ohta, S, etc. published a report entitled “Midkine is expressed during repair of bone fracture and promotes chondrogenesis” in the “Journal of Bone and Mineral Research” (1999(14):1132-1144), which finds that mouse ATDC5 cells transfected with MK gene can promote cartilage matrix synthesis. But the research also pointed out that not all high expression of MK-transfected cell line can produce cartilage matrix. In cultured ATDC5 cells, MK did not promote the synthesis of cartilage matrix. Dreyfus, J, etc. published a report entitled “HB-GAM/pleiotrophin but not RIHB/midkine enhances chondrogenesis in micromass” in the “Experimental Cell Research” (1998(241):171-180), which finds that, similarly, addition of exogenous MK can not promote mesenchymal cells from chicken embryos in micromass culture to synthesis cartilage matrix. These results demonstrate that MK does not promote chondrocyte growth and differentiate in vitro. However, the inventors discovered that MK promoted the growth of three different cartilage tissues in normal animals and that addition of exogenous MK protein to single layer culture promoted proliferation of the three types of chondrocytes without changing the expression profile of chondrocyte specific genes.